An application of nerve growth factor (NGF) to the dorsal root ganglion (DRG) neurons induces an elongation of axons. NGF activates the motility of growth cones locating at the tip of DRG axons. We synthesized a novel fluorescent analog of NGF, Cy3-NGF, to observe the signal input of NGF to the receptor of DRG growth cones. Conjugation of Cy3 to NGF was performed so that one molecule of Cy3 was cross-linked to the single molecule of NGF El Elsubunit. Excitation laser beam was diagonally illuminated to the growth cone to monitor the behavior of single molecules of fluorescent NGF on the surface of the growth cones. An application of 40pM Cy3-NGF to chick DRG neurons, which had been incubated in the solution without NGF, caused an extension of lamellipodia at the leading edge of the growth cones. Increase in the number of binding of Cy3-NGF to the surface of growth cone reached plateau phase after 10min from the application of NGF, when nearly 80% of the growth cones showed extension of
… Morelamellipodia. At this phase, the number of Cy3-NGF spots bound onto a single growth cone was counted to be 50-100. We found that there were two types of the behavior of Cy3-NGF molecules that had been bound to the receptors in the membrane of growth cones ; (1)two-dimensional diffusion with a diffusion constant of 0.2μm^2/s, which was slightly higher than that had been reported on PC12 cells estimated by the time course of fluorescent recovery after photo-bleaching of Rhodamine NGF, (2)one directional movement towards the center of the growth cone with a mean velocity of 0.08μm/s, which was close to that of constitutive rearward flow driven by actin cytoskeleton. This one directional movement of Cy3-NGF was blocked by a treatment of 200nM Latrunculin B, a drug that inhibits actin polymerization. The rearward movement of Cy3-NGF resulted in the accumulation of Cy3-NGF clusters at the central portion of the growth cone, where lots of membrane-bound vesicles were observed. The locations of some of the accumulated NGF clusters coincided with those of small vesicles. The two modes of movement of Cy3-NGF and its receptor complexes were discussed in terms of the mechanisms of NGF signaling in the growth cones. Less